Spinal and bulbar muscular atrophy is a neurodegenerative disease that affects lower motor neurons. Recent studies have shown that also muscle tissue is involved in the disease. In fact both motor neurons and muscle cells express the androgen receptor carrying the expanded polyQ. In presence of testosterone AR polyQ is released from the heat shock protein and undergoes conformational modification and possibly protein misfolding. Many misfolded AR aggregates are found in the nucleus of motor neuron and in the cytoplasm of dorsal root ganglia neurons and these are index of altered protein clearance.
The protein quality control (PQC) system is responsible for protein homeostasis in cells. Initially the heat shock protein system try to prevent protein misfolding. If refolding fails toxic misfolded protein are carried to the degradative systems: the ubiquitin proteasome system (UPS) and the autophagy. While proteasome is responsible for the degradation of protein with high turnover, autophagy removes high molecular weight aggregates by sequestering them into autophagosomes that will be fused to lysosomes for protein degradation. It has already been shown that improving autophagy leads to a reduction of aggregate in motor neurons. Since muscle is involved in the onset of the disease, here we study the involvement of the PQC system in muscle cells in presence of AR polyQ.
Using Filter Trap Assay (FTA), Western Blot (WB) and Immunofluorescence (IF) analyses we investigate which is the preferential pathway for the AR degradation in muscle C2C12 cells. We found that AR is mainly degradated trough UPS but autophagy is more involved in the degradation of the AR polyQ than wt AR.
Thus we potentiate autophagy using the mTOR independent activator trehalose. Using Real Time PCR we demonstrate that in our cells trehalose upregulate many genes involved in autophagic machinery like p62, LC3 and the Small Heat Shock Protein B8 (HspB8). We have previously showed that HspB8 is a potent autophagic facilitator. Then with FTA we demonstrated that trehalose reduces AR aggregates. HspB8 overexpression also reduces AR aggregation.
In conclusion we show that also in muscle cells AR aggregation can be reverted and we demonstrate that pharmacologically by activation of autophagy thus reducing the presence of the toxic misfolded protein. We demonstrated that modulation of the PQC system could be a promising pharmaceutical strategy to treat SBMA.
GRANTS: Regione Lombardia; AFM-TELETHON, FRANCE; FONDAZIONE TELETHON, ITALY; FONDAZIONE CARIPLO, ITALY; FONDAZIONE ARISLA, ITALY; Ministero della Sanità, ITALY.